Foudraine Dimard E, Dekker Lennard J M, Strepis Nikolaos, Nispeling Stan J, Raaphorst Merel N, Kloezen Wendy, Colle Piet, Verbon Annelies, Klaassen Corné H W, Luider Theo M, Goessens Wil H F
Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), Rotterdam, Netherlands.
Department of Neurology, Neuro-Oncology Laboratory, Clinical and Cancer Proteomics, Erasmus University Medical Center (Erasmus MC), Rotterdam, Netherlands.
Front Microbiol. 2022 Jun 22;13:887420. doi: 10.3389/fmicb.2022.887420. eCollection 2022.
New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the detection of β-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing or complex. Selected targets were the β-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, ANT(2 )-I, and APH(3')-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6')-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing or complex.
对于来自阳性血培养物的细菌，需要新的快速抗菌药物敏感性/耐药性检测方法。在本研究中，开发并验证了一种多重靶向液相色谱-串联质谱（LC-MS/MS）检测方法，用于检测生长缓慢或复杂的血培养物中β-内酰胺类、氨基糖苷类和氟喹诺酮类的耐药机制。选定的靶点包括β-内酰胺酶SHV、TEM、OXA-1样、CTX-M-1样、CMY-2样、染色体AmpC（cAmpC）、OXA-48样、NDM、VIM和KPC；氨基糖苷类修饰酶AAC(3)-Ia、AAC(3)-II、AAC(3)-IV、AAC(3)-VI、AAC(6')-Ib、ANT(2")-I和APH(3')-VI；16S核糖体甲基转移酶ArmA、RmtB、RmtC和RmtF；喹诺酮类耐药机制QnrA、QnrB、AAC(6')-Ib-cr；GyrA的野生型喹诺酮类耐药决定区；以及孔蛋白OmpC和OmpF。使用100份前瞻性收集的阳性血培养物以及148份添加了先前从血培养物中分离出的菌株或携带较少见耐药机制的菌株的阴性血培养物样本对所开发的检测方法进行评估。出结果的时间约为3小时。将LC-MS/MS结果与全基因组测序结果及抗菌药物敏感性检测结果进行比较。总体而言，LC-MS/MS结果与全基因组测序结果高度一致。此外，基于LC-MS/MS结果能够正确预测大多数敏感和非敏感表型。例外情况是环丙沙星和阿莫西林/克拉维酸的预测，分别与85.9%和63.7%的分离株表型相符。基于平行反应监测的靶向LC-MS/MS可用于快速、准确地检测生长缓慢或复杂的血培养物中的各种耐药机制。
